The largest amber-preserved flower revisited

Amber exquisitely preserves the delicate organs of fossil flowers for millions of years. However, flower inclusions can be rare and usually do not exceed 10 mm in size. Here we report an exceptionally large flower from late Eocene Baltic amber, measuring 28 mm across, which is about three times as large as most floral inclusions. This fossil was described over 150 years ago as Stewartia kowalewskii (Theaceae) and has never been revised. The analysis of pollen extracted from the anthers of the flower inclusion, however, revealed strong affinities to Asian species of Symplocos (Symplocaceae), prompting the new combination Symplocos kowalewskii comb. nov. et emend. This fossil represents the first record of Symplocaceae from Baltic amber and supports affinities of its flora to evergreen broadleaved and mixed mesophytic forests of present-day East and Southeast Asia. The rarity of such large-sized flower inclusions is likely due to the size of the resin outpouring and its properties, which might affect the embedding of plant organs.


Remarks.
The fossil was first published as Stewartia kowalewskii Casp. (Theaceae; also occasionally spelled Stuartia 20 ), but not figured and only briefly described as a well preserved pentamerous corolla of 28 mm in diameter with attached stamens 18,21 . Since then, the flower inclusion was frequently mentioned by various authors and occasionally figured over the last decades [22][23][24][25][26][27][28][29] . However, it was never documented in detail nor its identification thoroughly assessed. Kirchheimer 30 considered the fossil as similar to Stewartia I.Lawson but thought that the corolla did not provide sufficient evidence to demonstrate affinities to Stewartia. Affinities to the Theaceae, specifically the Camellioidae, were further suggested 31,32 but never unambiguously proven. Indeed, the inclusion resembles members of the Camellioideae (including Stewartia) in, for example, the basally connate and numerous (uncountable) stamens arranged in rows; the basifixed anthers (basifixed in some Camellia L. species, but dorsifixed in Stewartia) which lack an apical prolongation of the connective; the length of filaments, which are nearly as long as the petals; and the basally fused corolla [32][33][34][35][36] (Table 1). According to Tsou 37,38 , the only diagnostic character of the Camellioideae is the presence of pseudopollen in the connective of the anthers. We could not detect any pseudopollen in the anthers of the amber specimen. However, we are aware that despite the exquisite preservation of the amber specimen, the presence of such pseudopollen would be difficult to assess because it is rather small and inserted into the connective. The extracted pollen of the fossil shows distinct features of Symplocos Jacq. (Symplocaceae) as it exhibits: tricolporate apertures with short colpi (polar axes/colpus length ratio), oblate to subspheroidal shape with a triangular to circular outline in polar view and conspicuous vestibulum. Tectum sculpture and ornamentation is variable: densely verrucate, rugulate to verrucate, a combination of rugulate to microreticulate, perforate, microverrucate, and microreticulate with or without supratectal ornamentation 39 . Additionally, the combination of gross morphological characters is also indicative for Symplocos (Symplocaceae), including gamopetalous  (h) Pollen under electron microprobe, with exposed intine (arrowhead). (i,j) Pollen under electron microprobe, showing perforate to microreticulate ornamentation with few supratectal blunt echini. Note the fused verrucae at the margo of the ectoaperture, forming a rim-like structure (j). Scale bars 500 µm in (a); 50 µm in (b); 20 µm in (c-g); 10 µm in (h), 1 µm in (i,j). ) and Symplocos subgenus Symplocos are distinguished by characters that are mainly not preserved in the fossil, e.g., the numbers of carpels of the gynoecium. However, filaments of S. paniculata are terete and not constricted apically 40 , whereas they are flattened in the amber specimen and taper towards the anthers. Moreover, in contrast to the amber specimen, the pollen of S. paniculata is rather small (26-28 µm in diameter) and has a triangular to concave triangular outline in polar view. Furthermore, the pollen of S. paniculata is unique in the rugulate to microreticulate sculpture with perforations and fossulae in between the rugulae, producing a bireticulated pattern. Also as opposed to the amber specimen, pollen of S. paniculata has no supratectal ornamentation 43,44 .
Therefore, the amber specimen is more closely affiliated with Symplocos subgenus Symplocos. As based on phylogenetic analysis, this subgenus is divided into taxa (corresponding to clades), including Symplocos sections Barberina A.DC., Lodhra G.Don and Symplocos. The latter is divided into series Symplocos and Urbaniocharis (Brand) P.W.Fritsch 40 . The fossil can be excluded from sect. Symplocos based on the combination of its large size (thus excluding series Urbaniocharis, the species of which have corollas < 10 mm long 45 ), the non-monadelphous stamens (in series Symplocos, stamens are connate roughly halfway), and the androecial adnation merely at the base of the corolla (androecium is adnate about halfway to the corolla in series Symplocos). Moreover, within series Symplocos, the informal group (clade) "Neosymplocos" is distinguished from the fossil by its pubescent filaments 40 .
The remaining sections Barberina and Lohdra can only be effectively compared to the amber fossil on the basis of pollen morphology. About 86 extant Symplocos pollen species have been documented in the literature 41,42,44,46,47 . However, the documentation of sculpture variation of extant as well as fossil Symplocos pollen with SEM is incomplete because most pollen images are depicted only with light microscopy 48 . In comparing the available extant Symplocos pollen types with those from S. kowalewskii, only a few Asian species resemble the amber specimen in shape, size, outline and ektexine sculpture and supratectal ornamentation, namely S. obtusa Wall., S. pergracilis (Nakai) Yamazaki, S. tanakae Matsamura, and to a lesser extent S. pseudobarberina Gontscharow (all of S. section Lodhra). These species are all characterized by a perforate to microreticulate tectum and supratectal verrucae and occasional supratectal echini 40,42 , which is somewhat similar to S. kowalewskii. However, the density, number and sizes of these supratectal elements differ from those in S. kowalewskii and vary considerably among the named extant species (quite dense in S. tanakae, larger and more loose or regularly distributed in the other species).
In section Barberina, some resemblance occurs in the tectum of S. variabilis 49 ; however, the overall shape and the rounded apex of the colpus differ from the states of S. kowalewskii.
Among the fossil record, pollen of S. kowalewskii resembles two fossil Symplocos pollen types from the early Oligocene Haselbach locality (Germany 43 , Symplocos sp. 2 and sp. 8) in being microreticulate to foveolate or perforate with supratectal verrucae and baculae. As in S. kowalewskii, these pollen types bear similarities to the extant Asian species S. obtusa, S. pergracilis, S. tanakae and S. pseudobarberina.
All in all, the flower and pollen morphology of the amber inclusion is indicative enough to justify its assignment to Symplocos subgenus Symplocos with the new combination Symplocos kowalewskii (Casp.) Sadowski 55 .
In Symplocos bureauana and S. subspicata, the gynoecium was not preserved and pollen extraction from the fossils was unsuccessful 55 . In comparing both species with S. kowalewskii (Table 1) Table 1). Thus, features of S. kowalewskii clearly differentiate it from other fossil taxa, justifying its treatment as a distinct species. Among the numerous flower inclusions from Baltic amber 18 , none shows the same set of indicative features as S. kowalewskii. Thus, S. kowalewskii is the first fossil record of this genus and of the Symplocaceae from Baltic amber.

Palaeoecological implications. Symplocaceae from the early Eocene flourished in paratropical forests
with deciduous and evergreen taxa and multilayered canopies (e.g. early Ypresian, Fisher/Sullivan site, Virginia, United States 51,62 ). In younger fossil floras, Symplocaceae also dominated forested areas (e.g. in Miocene of Vogelberg/Salzhausen, Germany 55 ) or grew in the understory of lowland hinterland forests, mixed with conifers and angiosperms (middle Miocene, Lavantal Basin, Austria 48 ; late early Miocene, Wiesa, Germany 53,63 ). In the early Oligocene Haselbach horizon (Leipzig Embayment, Germany 64 ), Symplocos species were one of the main constituents of mixed mesophytic forests, but also occurred in Quasisequoia swamp forests 65 . Most species of extant Symplocaceae are evergreen shrubs and trees that grow from 500 up to 4000 m elevation of tropical zones, being most abundant in mountain forests of 2500-3500 m elevation 36 . The fossil and extant occurrences of Symplocaceae indicate that the family thrives in humid mixed-mesophytic forests in warm-temperate to subtropical climates, whereas arid regions are avoided 31,44,48,[66][67][68] .
This agrees with the most recent analyses of the Baltic amber source area, the so-called Baltic amber forest, where humid and warm-temperate conditions likely prevailed. Furthermore, assessment of inclusions of hyperdiverse Fagaceae and conifers, as well as fungi and lichens, show that the Baltic amber forest was heterogeneous, including coastal swamps, bogs, riparian forests and mixed conifer-angiosperm forests intermingled with open areas 7,15,16,[69][70][71][72] . As indicated by the fossil record of Symplocaceae, Symplocos kowalewskii was likely part of the forested habitats in the Baltic amber source area. As known from the Oligocene Haselbach flora, it is also possible that S. kowalewskii was associated with Quasiseuqoia swamps, since this conifer has recently been confirmed from Baltic amber 15 66 . Not before the Pliocene, Symplocaceae dispersed from Europe to eastern Asia, as it was indicated by its macrofossil record 66,68 . However, recently discovered fossil pollen of Symplocos subgen. Palura from the middle Eocene of Hainan 44 and Symplocos pollen from the Paleogene of the far East of Russia (named "Proteacidites" 73 ) have challenged notions about the Paleogene distribution of Symplocaceae and indicate that the family was already present in Asia by that time.
As discussed above, Symplocos kowalewskii and its pollen shows the most similarities to extant species of Asia. Symplocos obtusa is found in South India and Ceylon (at elevations of 1800-2400 m 33 Flower size and preservation of Symplocos kowalewskii. Among extant Symplocaceae, corollas generally range in size between 3 and 13 mm 33,35 , but also can become larger 76 . Flower size is apparently without consistent taxonomic significance 40 . Nonetheless, corolla size and the degree of petal fusion can be an indicator of different pollination syndromes. For example, the androecium of flowers in most of the species of Neotropical Symplocos sect. Symplocos is distally more adnate to the tubular-shaped corolla. This is interpreted as adaptation to pollination by long-tongued bees and long-billed hummingbirds that are able to reach the copious nectar 40,77 . In contrast, most Asian species of Symplocos are thought to be generally insect pollinated, which would explain why their corollas and androecia are lesser fused than in Neotropical Symplocos sect. Symplocos 40 . The corolla of S. kowalewskii is only basally fused, as is the case for many extant insect-pollinated Symplocaceae. An additional indication for insect pollination of S. kowalewskii might be the unusually large corolla that likely served as attraction to insect pollinators 78 . The exceptional preservation of amber inclusions like S. kowalewskii is possibly caused by the biocidal properties of the embedding resin, which inhibits degradation processes 79,80 . In many plant inclusions, internal and external structures are three-dimensionally preserved 13 , similar to mummifications 16 ; however, it is unknown as to which processes are involved in the preservation of plants in amber. In contrast, the taphonomy of animal inclusions has been studied in detail, showing numerous factors that control their preservation, such as size of the organism, resin viscosity and stickiness [80][81][82][83] . Some of these factors could be similarly important for plant inclusions. Depending on the resin surface tension and viscosity, smaller plant organs are likely more easily retained than larger ones. Based on the known plant inclusions, it is evident that there is a size limitation in amber (Supplementary Table S1). This is probably also related to the size of the resin trap; in Baltic amber only 4.7% of the mined pieces are > 32 mm in size whereas more than 40% measure < 18 mm 84 , showing that larger pieces, as in this study, are rare. In considering the overall scarcity of plant inclusions 9,10 , as well as the taphonomical biases, amber inclusions like Symplocos kowalewskii are unique in preservation and size.
Additionally, the flower of S. kowalewskii was trapped by the resin during anthesis, providing enough mature pollen for extraction, which is also a rather rare condition in flower inclusions 85 . The amber pollen of S. kowalewskii exhibits exceptional preservation as well; for example, SEM analyses revealed preservation of the pollen wall, including the intine (Fig. 3h), which is normally lacking after preparing conventional palynological material 86 . Furthermore, the well-preserved details of the pollen ornamentation facilitated the identification of the flower, which shows the great benefit of pollen extraction, as well as the necessity of SEM analyses.
Some pollen of the amber specimen show protrusion-like structures at the apertures (Fig. 3c), which likely were caused by the excretion of cellular content. This is somewhat similar to the effect of acetolysis where the cellular content of the pollen is dissolved and excreted. It seems likely that the fresh resin had a similar effect on some pollen, but the chemical processes that might have caused this effect during embedding or amberization are unknown.

Conclusion
Symplocos kowalewskii (Symplocaceae) from Baltic amber is the by far largest flower inclusion known. Its in-situ pollen, combined with morphology of the corolla and androecium, indicates strong affinities to extant Asian species of S. subgen. Symplocos. The large size of the corolla and its basal fusion to a staminate ring likely indicates entomophilous pollination, as is known for some Asian Symplocaceae. S. kowalewskii was likely a constituent of mixed-angiosperm-conifer forests in the Baltic amber source area and supports its affinities to evergreen broadleaved and mixed mesophytic forests of present-day East and Southeast Asia.

Material and methods
Origin and age of the amber fossil. The  www.nature.com/scientificreports/ therein). The so-called Blue Earth layer yields the highest amounts of amber and is therefore mainly subjected to mining [87][88][89] . Baltic amber is also occasionally found along the Baltic Sea coast, mostly being eroded from the Blue Earth that is exposed along the coast of the Samland Peninsular 90 . Different age estimates of the Blue Earth layer and its amber have been suggested, ranging from an early Eocene 91 to late Eocene age [89][90][91]  Preparation, imaging and pollen analysis. The amber specimen X4088 was enclosed in a glass chamber, filled with a solution of an extant dammar resin (Shorea, Dipterocarpaceae) and covered with a cover slip. This kind of preparation is typically found in historical Baltic amber collections that were established in Königsberg (today Kaliningrad, Russia) during the late 19th to early twentieth century ( 95 and references therein). In X4088 the glass chamber limited viewing details of the flower inclusion and inhibited pollen extraction. Moreover, the glass chamber and the cover slip were partially fractured and the embedding medium showed several signs of deterioration, including yellowing and fissures (Fig. 1a,b). We heated the specimen gently for five minutes at 38 °C in a vacuum oven (VO200, Memmert), which liquefied the embedding medium and allowed us to remove the fractured parts of the cover slip. As the resin cooled and began to cure, the amber specimen was again heated at 38 °C for several minutes. Then, the viscous resin was scratched out of the glass chamber by using a wooden pick and a scalpel. The remaining thin layer of sticky Dammer resin was ground and polished away by using wet silicon carbide paper (manufacturer Struers; see 95 for protocols). A wet leather cloth with a tooth paste suspension was used for polishing all amber surfaces. The amber inclusion was studied under a binocular microscope (Stemi 508, Carl Zeiss), a dissecting microscope (StereoDiscovery V8, Carl Zeiss) and a light microscope (AxioScope A1 KMAT, Carl Zeiss). Image stacks were taken with digital cameras (Canon EOS 80D), that were installed on each microscope, and further processed into photomicrographic composites by applying HeliconFocus. Up to nine singular photomicrographic composites were merged with the Adobe Photoshop CS6 23.0.0 software to create overview images of Fig. 1d,e. The overview images of Fig. 1a-c were taken using a photo station and a digital camera (Sony ILCE 7RM3), equipped with a Sony FE 50 mm F2.8 Macro Lens and the computer software Imaging Edge Desktop.
Pollen was carefully scratched out of one anther and the surrounding amber with a scalpel. We placed the samples on carbon-covered Scanning electron microscopy (SEM) mounts and sputtered them with gold (6 nm coat thickness) using an Automatic Sputter Coater. The pollen sample was examined under an electron microprobe with a field emission cathode (JEOL JXA-8500F).
For comparing the amber specimen to other fossil flowers of Symplocos, we used descriptions in the literature (as given in Table 1). For S. bureauana, microscopic images of the holotype MNHN.F-2170.2 (Paleontology collection, Muséum national d'Histoire naturelle, Paris, France; MNHN) helped to estimate the size of the flower organs.

Data availability
The fossil specimen is part of the public collection of the Federal Institute for Geosciences and Natural Resources (Bundesanstalt für Geowissenschaften und Rohstoffe, BGR) in Berlin-Spandau (Germany). All data generated or analysed during this study are included in this published article.